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Biomedical subjects

R A Roth

Publications and source records attributed to R A Roth.

At least 235 records · Page 13Linked to original sources
PubMed1987

Defective internalization of insulin and its receptor in cells expressing mutated insulin receptors lacking kinase activity.

The internalization and degradation of insulin was assessed in Chinese hamster ovary cell lines expressing either the wild-type receptor or mutated receptors lacking kinase activity. The mutated receptors included receptors which differed from the wild-type receptor by a single amino acid (substitution of an arginine for lysine at position 1030, a site critical for ATP binding) as well as receptors which had a deletion of 112 amino acids at the carboxyl terminus. Cells expressing mutated receptors lacking kinase activity were found to internalize and degrade insulin at about half the rate of cells expressing wild-type receptors with kinase activity. Moreover, insulin was found incapable of inducing the internalization of the mutated receptors, whereas it could stimulate the internalization of the wild-type receptor. Finally, the constitutive rate of receptor internalization was found to be the same for the mutant and wild-type receptors. These results implicate the intrinsic tyrosine-specific kinase activity of the insulin receptor in the ligand-induced, but not the constitutive, internalization of this receptor.

Animals↗
PubMed1987

In vivo cross-linking of protein disulfide isomerase to immunoglobulins.

To test the proposed role of protein disulfide isomerase in the synthesis of immunoglobulins (Ig), intact lymphocytes were treated with a thiol-cleavable, bifunctional cross-linking agent and lysed, and the lysates were immunoprecipitated with antibodies to either Ig or enzyme. When the immunoprecipitates were analyzed on polyacrylamide-sodium dodecyl sulfate gels, protein disulfide isomerase was found to be cross-linked to immunoglobulins. The extent of cross-linking was dependent upon the concentration of cross-linker added and the class of Ig. For IgMs and high concentrations of cross-linker, approximately one molecule of Ig was coupled per two molecules of enzyme. For IgGs, the extent of cross-linking was less. Finally, depletion of the intracellularly reduced glutathione by diamide was found to also result in the linkage of protein disulfide isomerase to IgM. These results therefore support the hypothesis that protein disulfide isomerase functions in the in vivo synthesis of immunoglobulins.

Animals↗
PubMed1987

Antibody-induced down-regulation of a mutated insulin receptor lacking an intact cytoplasmic domain.

Insulin receptor down-regulation was studied in various Chinese hamster ovary (CHO) cell lines expressing transfected human insulin receptor cDNAs. In addition to a cell line expressing the normal receptor (CHO.T line), three lines expressing mutated receptors were studied: the CHO.T-t line, which expresses a receptor with a degraded cytoplasmic domain due to the removal of the C-terminal 112 amino acids, and the CHO.YF1 and CHO.YF3 lines, in which important autophosphorylation sites of the receptor kinase (tyrosines-1162 and -1163) have been replaced by phenylalanine. A monoclonal anti-receptor antibody, but not insulin itself, was found to down-regulate cell surface receptor levels in all four cell lines by 60-80% after 18-h treatment at 37 degrees C. Down-regulation of the CHO.T and CHO.T-t receptors occurred at similar antibody concentrations and with a similar time course, although the maximum level of CHO.T-t down-regulation (60%) was generally lower than the level of CHO.T down-regulation (80%). Pulse-chase labeling of these two cell types with [35S]methionine revealed that antibody treatment of both CHO.T and CHO.T-t cells resulted in a similar increase in the rate of degradation of mature receptor subunits. These results indicate that antibody-induced down-regulation of the insulin receptor in these cells can occur in the absence of various autophosphorylation sites of the receptor and that the mechanism of antibody-induced down-regulation is different from that for insulin.

Animals↗
PubMed1987

Ras p21 as a potential mediator of insulin action in Xenopus oocytes.

The oncogene protein product (p21) of the ras gene has been implicated in mediating the effects of a variety of growth factors and hormones. Microinjection of monoclonal antibody 6B7, which is directed against a synthetic peptide corresponding to a highly conserved region of p21 (amino acids 29 to 44) required for p21 function, specifically inhibited Xenopus oocyte maturation induced by incubation with insulin. The inhibition was dose-dependent and specific since (i) the same antibody had no effect on progesterone-induced maturation, (ii) immunoprecipitation and Western blotting indicated that the antibody recognized a single protein of molecular weight 21,000 in oocyte extracts, and (iii) inhibition was not observed with identical concentrations of normal immunoglobulin. Thus, p21 appears to be involved in mediating insulin-induced maturation of Xenopus oocytes. Furthermore, the mechanism may involve phosphorylation of p21, as p21 was found to be a substrate of the insulin receptor kinase.

Animals↗
PubMed1987

The receptor for insulin-like growth factor II mediates an insulin-like response.

Insulin-like growth factor II (IGF-II) shares sequence homology and predicted three-dimensional structure with insulin and IGF-I. IGF-II can bind, therefore, to a limited extent with the receptors for these two other hormones, as well as to a distinct receptor for IGF-II. Previous studies have been unable to attribute a particular response of IGF-II through its own receptor. In the present studies, the IGF-II receptor is shown to mediate the stimulation of glycogen synthesis in human hepatoma cells since: (i) IGF-II is found to be capable of stimulating a response at concentrations in which it would primarily interact with its own receptor; (ii) the response to IGF-II was not blocked by monoclonal antibodies which inhibit the responses of cells through the insulin and IGF-I receptors; and (iii) polyclonal antibodies to the IGF-II receptor were found to mimic the ability of IGF-II to stimulate glycogen synthesis. These results indicate that the IGF-II receptor mediates a particular biological response--stimulation of glycogen synthesis in hepatoma cells. Furthermore, a monovalent Fab fragment of the polyclonal antibody to the IGF-II receptor was also shown to stimulate glycogen synthesis in these cells. These data indicate that clustering of the IGF-II receptor is not required to stimulate a biological response.

Antibodies, Monoclonal↗
PubMed1987

5-Hydroxytryptamine and thromboxane in platelets from rats treated with monocrotaline pyrrole.

Monocrotaline pyrrole (MCTP), a metabolite of the plant toxin monocrotaline, produces pulmonary vascular injury, pulmonary hypertension, and right ventricular enlargement (RVE) in rats by an unknown mechanism. A role for platelets has been suggested by the observation that antibody-induced thrombocytopenia reduces the RVE caused by MCTP. The platelet can release a number of vasoconstrictive agents, such as 5-hydroxytryptamine (5HT) and thromboxane A2 (TxA2), that could possibly contribute to pulmonary hypertension. It was of interest to determine whether treatment with MCTP alters platelet 5HT content or alters the release of TxA2 in platelet-rich plasma (PRP) in response to aggregation. Fourteen days following treatment with MCTP when pulmonary hypertension is well-established and RVE is present, the concentration of 5HT in washed platelets or in platelet-poor plasma was not different in treated and control rats. One day following treatment with MCTP, before lung injury is evident, the concentration of TxB2, a stable metabolite of TxA2, was higher in unstimulated PRP from treated rats than in control rats. The concentration of TxB2 was also examined in PRP at 4 days (when lung injury first appears), 7 days (when pulmonary arterial pressure first increases), and 14 days after treatment with MCTP (when RVE is evident). At 4, 7, or 14 days following treatment there was no difference in the concentration of TxB2 in unstimulated PRP from MCTP-treated and control rats. Following stimulation with arachidonic acid, the release of TxB2 at maximal aggregation was not different in PRP from MCTP-treated and control rats at any time after treatment. The rate of release of TxB2 was lower in PRP from rats treated with MCTP 7 days earlier, but was not different at any other time following treatment. At concentrations up to 250 micrograms/ml, MCTP added in vitro to PRP from untreated rats did not affect the concentration of TxB2 released during aggregation induced by arachidonic acid. Only at very high concentrations (1 mg/ml) did MCTP abolish the aggregation response and depress TxB2 release in PRP. These results indicate that MCTP treatment does not affect platelet 5HT content and does not affect basal TxB2 production or TxB2 release by platelets stimulated in vitro.

Animals↗
PubMed1987

The effect of immunosuppressants and adoptive transfer in monocrotaline pyrrole pneumotoxicity.

Monocrotaline pyrrole (MCTP) is a pyrrolizidine alkaloid that causes pulmonary vascular injury and pulmonary hypertension in rats. The lesions in lungs of MCTP-treated rats are similar to those occurring in humans with primary pulmonary hypertension. Thus, the MCTP-treated rat is a good animal model for this disease. The mechanisms by which MCTP causes lung injury are unknown. The character of the pulmonary lesions and the delay in onset of the injury after a single low dose of MCTP suggest that immune mechanisms may be important in the pathogenesis. Accordingly, rats were treated with MCTP and the immunosuppressants antilymphocyte serum (ALS) or cyclosporin A (CyA). Neither ALS nor CyA completely protected rats from the injury due to MCTP. Several series of experiments also were undertaken to assess the effect of lymphocytes adoptively transferred from MCTP-treated donor rats into MCTP-treated recipient rats. Adoptive transfer of lymphocytes did not decrease the onset time of the injury or increase the severity of lesions due to MCTP in the recipients. These results indicate that immune mechanisms are probably not involved in MCTP-induced pulmonary injury.

Animals↗
PubMed1987

Phorbol myristate acetate produces injury to isolated rat lungs in the presence and absence of perfused neutrophils.

Because lung injury induced by phorbol myristate acetate (PMA) has been reported by some to be mediated by blood neutrophils (PMN) and by others to occur independently of PMN, we examined the PMN dependency of PMA-induced injury in isolated, perfused lungs of rats. Depending on dose, PMA added to medium perfusing isolated rat lungs produced injury in the presence or in the absence of added PMN. When a high concentration of PMA (57 ng/ml) was added to medium devoid of added PMN, perfusion pressure and lung weight increased. Superoxide dismutase (500 U/ml) and catalase (400 U/ml) added to the medium prior to PMA had no effect on the increases in lung weight or perfusion pressure. When a concentration of PMA (21 ng/ml or less) that did not by itself cause lungs to accumulate fluid was added to perfusion medium containing PMN (1 X 10(8)), superoxide was produced, perfusion pressure increased, and lungs accumulated fluid. Addition of superoxide dismutase and catalase to this preparation prevented the increase in lung weight, but not the increase in perfusion pressure. We conclude that high concentrations of PMA produce lung injury which is independent of neutrophils and oxygen radicals and that lower concentrations produce injury which is neutrophil-dependent and mediated by oxygen radicals. These results may explain why PMA-induced lung injury has variously been reported to be PMN-dependent in some systems and PMN-independent in others.

Animals↗
PubMed1987

Involvement of thromboxane in injury to isolated rat lungs perfused with phorbol myristate acetate in the presence and absence of neutrophils.

In a previous study, we demonstrated that a non-toxic concentration of phorbol myristate acetate (PMA) produced edema in isolated rat lungs which were coperfused with neutrophils (PMN). In this study, we examined whether prostaglandins or thromboxane were responsible for increases in pressure and/or edema in this preparation. In lungs perfused with PMA (14 ng/ml) and PMN (1 X 10(8], significantly greater amounts of thromboxane B2 (TxB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) were produced than in controls. Relative lung weights and increases in perfusion pressure correlated with concentrations of TxB2 and 6-keto-PGF1 alpha that were produced. Indomethacin (10 microM) or Dazmegrel (10 microM) retarded the increase in perfusion pressure and prevented the increase in relative lung weight induced by PMA and PMN. When lungs were perfused with a high concentration of PMA (57 ng/ml) in the absence of added PMN, lungs also become edematous. Compared to controls, concentrations of TxB2 and 6-keto-PGF1 alpha were elevated in media collected from this preparation. As with lungs perfused with PMN and PMA, increases in pressure and relative weights of lungs perfused with PMA (57 ng/ml) correlated with the concentrations of TxB2 that were detected in perfusion media. Although indomethacin (10 microM) and Dazmegrel (50 microM) retarded the increase in perfusion pressure in this preparation, they only partially attenuated the increase in lung weight. These results suggest that, depending on the concentration, PMA can produce lung injury via different mechanisms. Thromboxane does not seem to be required for the genesis of edema induced by a high concentration of PMA in the absence of perfused neutrophils; however, it appears to play an obligatory role in the pathogenesis of edema induced by a low concentration of PMA in the presence of PMN.

6-Ketoprostaglandin F1 alpha↗
PubMed1987

Acute insulin action requires insulin receptor kinase activity: introduction of an inhibitory monoclonal antibody into mammalian cells blocks the rapid effects of insulin.

The role of the insulin receptor tyrosine kinase (protein-tyrosine kinase, EC 2.7.1.112) in various rapid insulin effects was studied by injecting four different cell types (by osmotic lysis of pinocytotic vesicles) with a monoclonal antibody that specifically inhibits the kinase activity of the insulin receptor and the closely related receptor for insulin-like growth factor (IGF)-I. Injection of this inhibitory antibody resulted in a decreased ability of insulin to stimulate the uptake of 2-deoxyglucose in Chinese hamster ovary cells and freshly isolated rat adipocytes, ribosomal protein S6 phosphorylation in CHO cells, and glycogen synthesis in the human hepatoma cell line HepG2. The ability of insulin, IGF-I, and IGF-II to stimulate glucose uptake in TA1 mouse adipocytes was also inhibited. Studies with CHO cells demonstrated that these effects of the inhibitory antibody were specific, since there was no change in phorbol ester-stimulated glucose uptake and injection of a noninhibiting antibody to the kinase had no effect on insulin action. These studies indicate that the tyrosine kinase activity of the insulin receptor is important in mediating several rapid insulin effects in a variety of different cell types.

Adipose Tissue↗
PubMed1987

Heterologous transmembrane signaling by a human insulin receptor-v-ros hybrid in Chinese hamster ovary cells.

A hybrid receptor molecule composed of the extracellular ligand-binding domain of the human insulin receptor and the transmembrane and cytoplasmic (protein-tyrosine kinase) domains of the chicken sarcoma virus UR2 transforming protein p68gag-ros has been constructed and expressed in Chinese hamster ovary (CHO) cells. The hybrid is processed normally into alpha and hybrid beta subunits, is expressed on the cell surface at high levels, and binds insulin with near-wild-type affinity. Furthermore, insulin stimulates the phosphorylation on tyrosine residues of the hybrid beta subunit in vivo and the phosphorylation of an exogenous substrate [poly(Glu,Tyr)] in vitro. Thus the hybrid is capable of heterologous transmembrane signaling. However, the hybrid mediates neither the insulin-activated uptake of 2-deoxyglucose nor the incorporation of [3H]thymidine into DNA, suggesting that the physiological response(s) mediated by ligand-activated protein-tyrosine kinases may utilize distinct intracellular mechanisms for postreceptor signaling.

Animals↗
PubMed1987

Activation of transforming potential of the human insulin receptor gene.

A retrovirus containing part of the human insulin receptor (hIR) gene was constructed by replacing ros sequences in the avian sarcoma virus UR2 with hIR cDNA sequences coding for 46 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains of the beta subunit of hIR. The resulting virus, named UIR, contains the hIR sequence fused to the 5' portion of the UR2 gag gene coding for p19. UIR is capable of transforming chicken embryo fibroblasts and promoting formation of colonies in soft agar; however, it does not form tumors in vivo. A variant that arose from the parental UIR is capable of efficiently inducing sarcomas in vivo. UIR-transformed cells exhibit higher rates of glucose uptake and growth than normal cells. The 4-kilobase UIR genome codes for a membrane-associated, glycosylated gag-hIR fusion protein of 75 kDa designated P75gag-hir. P75gag-hir contains a protein tyrosine kinase activity that is capable of undergoing autophosphorylation and of phosphorylating foreign substrates in vitro; it is phosphorylated at both serine and tyrosine residues in vivo.

Animals↗
PubMed1987

Replacement of lysine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody-stimulated glucose uptake and receptor kinase activity.

To test whether the tyrosine kinase activity of the insulin receptor is crucial for insulin action, we have constructed mutations of the human insulin receptor at Lys-1030, which is in the presumed ATP-binding region. By using oligonucleotide-directed mutagenesis, this lysine residue was replaced with either methionine, arginine, or alanine. Chinese hamster ovary cells were transfected by mutant cDNAs and the expressed insulin receptors were characterized. We show here that none of these mutants exhibited insulin-activated autophosphorylation and kinase activity in vitro. They also do not mediate insulin- and antibody-stimulated uptake of 2-deoxyglucose. The tyrosine kinase activity is thus required for a key physiological response of insulin.

Adenosine Triphosphate↗
PubMed1987

Lack of effect of deferoxamine, dimethyl sulfoxide, and catalase on monocrotaline pyrrole pulmonary injury.

Monocrotaline pyrrole (MCTP) is a reactive metabolite of the pyrrolizidine alkaloid monocrotaline. MCTP given intravenously to rats causes pulmonary hypertension and right ventricular hypertrophy. Lesions in lungs after MCTP treatment contain macrophages and neutrophils, which may contribute to the damage by generation of reactive oxygen metabolites. Rats were treated with MCTP and agents known to protect against oxygen radical-mediated damage in acute models of neutrophil-dependent lung injury. Rats received MCTP and deferoxamine mesylate (DF), dimethyl sulfoxide (DMSO), or polyethylene glycol-coupled catalase (PEG-CAT). MCTP/vehicle-treated controls developed lung injury manifested as increased lung weight, release of lactate dehydrogenase into the airway, and sequestration of 125I-labeled bovine serum albumin in the lungs. Cotreatment of rats with DF, DMSO, or PEG-CAT did not protect against the injury due to MCTP. These results suggest that toxic oxygen metabolites do not play an important role in the pathogenesis of MCTP-induced pulmonary injury.

Animals↗
PubMed1987

Secondary ureteropelvic junction stricture: percutaneous dilation.

Percutaneous incision and balloon catheter dilation of the ureteropelvic junction (UPJ) was performed in nine patients with secondary UPJ strictures. In all patients, the UPJ was successfully dilated, and no immediate complications were encountered. After a postoperative follow-up period of 4-27 months, six patients showed marked improvement on excretory urograms. Three patients had recurrent obstruction after 3-18 months. The 66% success rate of percutaneous balloon dilation with or without endopyelotomy compares favorably with the success rate of open surgical repair of secondary UPJ strictures in adults.

Adult↗
PubMed1987

Thromboxane does not mediate pulmonary vascular response to monocrotaline pyrrole.

The possible involvement of thromboxane (Tx) in the pulmonary hypertension caused by monocrotaline pyrrole (MCTP) administration to rats was investigated by pharmacological intervention of Tx synthesis and action. The cyclooxygenase inhibitor, ibuprofen, at doses that inhibited platelet function and suppressed plasma Tx levels, did not attenuate MCTP-induced right ventricular hypertrophy or increased lung weight. Dazmegrel, an inhibitor of Tx synthetase, did not affect the MCTP-induced increase in lung weight or the elevation of lactate dehydrogenase activity or protein concentration in bronchopulmonary lavage fluid, despite significant reduction of the plasma concentration of Tx. Dazmegrel also did not alter the vascular leak or right ventricular hypertrophy due to administration of MCTP to rats. Finally, the Tx receptor antagonist L-640,035 was tested using a dosing regimen that reduced the increase in right ventricular pressure caused by a stable endoperoxide analogue in MCTP-treated rats. Cotreatment with L-640,035 did not attenuate the increase in lung weight, lavage fluid lactate dehydrogenase activity or protein concentration, or the pulmonary hypertension caused by MCTP. These results indicate that interference with Tx synthesis or action does not attenuate the toxic effects of MCTP and suggest that Tx is not necessary for the cardiopulmonary response to MCTP.

Animals↗